Rigidized trimethine cyanine dyes

ABSTRACT

Disclosed are analogues of trimethine cyanine dyes which are useful for imparting fluorescent properties to target materials by covalent and non-covalent association. The compounds have the following general formula:  
                 
 
     optionally substituted by groups R 2 -R 9  wherein groups R 6 , R 7 , R 8  and R 9  are attached to the rings containing X and Y or, optionally are attached to atoms of the Z a  and Z b  ring structures and groups R 1 -R 9  are chosen to provide desired solubility, reactivity and spectral properties to the fluorescent compounds;  
     A is selected from O, S and NR 11  where R 11  is the substituted amino radical:  
                 
 
     where R′ is selected from hydrogen, a C 1-4  alkyl and aryl and R″ is selected from C 1-18  alkyl, aryl, heteroaryl, an acyl radical having from  2 - 7  carbon atoms, and a thiocarbamoyl radical;  
     Z a  and Z b  each represent a bond or the atoms necessary to complete one, two fused or three fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms.

[0001] The present invention relates to rigidized trimethine cyaninedyes, their preparation, their use as fluorescent markers and influorescence energy transfer complexes and to materials labelled withthem.

[0002] Fluorescent dyes are generally known and used for fluorescencelabelling and detection of various biological and non-biologicalmaterials by procedures such as fluorescence microscopy, fluorescenceimmunoassay and flow cytometry. A typical method for labelling suchmaterials with fluorescent dyes is to create a fluorescent complex bymeans of bonding between suitable groups on the dye molecule andcompatible groups on the material to be labelled. In this way, materialssuch as cells, tissues, amino acids, proteins, antibodies, drugs,hormones, nucleotides, nucleic acids, lipids and polysaccharides and thelike may be chemically labelled and detected or quantitated, or may beused as fluorescent probes which can bind specifically to targetmaterials and detected by fluorescence detection methods.

[0003] Four commonly used classes of fluorescent dyes are those based onthe fluorescein (green fluorescence), rhodamine (orange fluorescence),coumarin and pyrene (blue fluorescence) chromophores. Dyes based onfluorescein and rhodamine have a number of disadvantages. Fluoresceinderivatives have a pH-sensitive absorption spectrum and fluorescenceyield decreases markedly below pH 8. Rhodamine derivatives arehydrophobic and are difficult to use in aqueous media. They often showstrong fluorescence quenching when bound to proteins.

[0004] U.S. Pat. No. 5,268,486 discloses luminescent mono- andpolymethine cyanine dyes and related polymethine dyes such asmerocyanine and styryl dyes which contain groups enabling them tocovalently attached to amine, hydroxyl, aldehyde and sulphydryl groupson a target material. The compounds are disclosed as fluorescing in thegreen, orange, red and near infra-red regions of the spectrum.

[0005] U.S. Pat. No. 3,679,427 describes rigidized cyanine dyes whichcontain a trimethine chain as part of a rigid structure, as shown informula (1):

[0006] where each of Z and Z¹ represents the non-metallic atomsnecessary to complete a heterocyclic nucleus of the type used in cyaninedyes; R represents a member selected from a hydrogen atom, an alkylradical, or an aryl radical; R¹ represents a member selected fromoxygen, sulphur, selenium or nitrogen. The subject dyes are reported toexhibit strong fluorescence and are useful spectral sensitizing dyes forphotographic silver halide as well as being useful as colorant materialsfor a wide variety of compositions such as paints, lacquers, etc.However they are not described as fluorescent labelling dyes.

[0007] European Patent Application No.747448 describes bis-heterocyclicmonomethine cyanine dyes, rigidized by means of a bridging group betweenthe nitrogen atoms of the heterocycles. Such compounds may besubstituted with additional groups chosen to provide desirablesolubility, reactivity and spectroscopic properties to the fluorescentcompounds. The dyes can be used to covalently label a target material soas to impart fluorescent properties to that target. The monomethinerigidized cyanines are highly fluorescent and strongly light-absorbingdyes which emit in the near UV and blue (300-500 nm) region of thespectrum. None of the foregoing literature discloses fluorescentrigidized dye compounds that are capable of producing strongfluorescence in the green to orange region of the spectrum and alsocontain functional groups and/or solubilizing groups which render thedye suitable for covalent labelling, in particular to biologicalmolecules and other target materials.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 is a graph showing the absorption spectra for a solution ofthe dye of Example 1 as compared to protein labeled with the dye inExample 1.6;

[0009]FIG. 2 compares the rigidized dye structure of the dye of Example1 with the structures of two open chain dyes.

[0010]FIG. 3 is a graph comparing the spectral properties of the threecyanine 3 dyes of FIG. 2;

[0011]FIG. 4 is a graph showing the emission spectra of the threecyanine dyes of FIG. 2 when excited at 514 nm;

[0012]FIG. 5 shows the results of photo bleaching the three dyes of FIG.2 when exposed to laser;

[0013]FIG. 6 is a graph showing the result of a peptide polarizationbinding assay according to Example 12.2;

[0014]FIG. 7 is a graph showing the results of the nucleic acid FREThybridization assay of Example 13.3;

[0015]FIG. 8 is a bar graph showing the results of a protein: DNA directintensity binding assay according to the procedures of Example 14.2;

[0016]FIG. 9 is a bar graph showing the results of a protein: DNA FRETbinding assay of Example 15.2; and

[0017]FIG. 10 is a graph plotting specific polarization readings againstconcentration of unlabeled ligand following Example 16.2.

[0018] The present invention provides bright, highly fluorescent dyecompounds which absorb and emit in the 450-600 nm region of thespectrum. They have rigid structures which are based on the trimethinecyanine chromophore and confer high quantum yields of fluorescence.Moreover, they can contain functional or reactive groups which may beused to covalently react with suitable groups on target materials suchas biological molecules, and other materials. They are pH insensitiveand thus they extend the range of useful fluorescent labelling reagentswhich can be used in fluorescent detection applications.

[0019] Accordingly, the present invention provides compounds of formula(2):

[0020] optionally substituted by groups R²-R⁹, wherein groups R⁶, R⁷, R⁸and R⁹ are attached to the rings containing X and Y or, optionally areattached to atoms of the Z^(a) and Z^(b) ring structures;

[0021] R² to R⁹ which are the same or different include —R¹ and —L—R¹⁰where R¹⁰ is selected from neutral groups that reduce water solubility,polar groups that increase water solubility, functional groups that canbe used in labelling reactions, reactive groups, electron donating andwithdrawing groups that shift the absorption and emission wavelengths ofthe fluorescent molecule, lipid and hydrocarbon solubilising groups, andL is selected from the group consisting of a straight or branched C₁₋₂₀alkyl chain, a C₂₋₂₀ monoether or polyether and a C₂₋₂₀ atom chaincontaining up to four secondary amide linkages;

[0022] R¹ is selected from hydrogen, aryl, heteroaryl, cyano, nitro,aldehyde, halogen, hydroxy, amino, quaternary amino, acetal, ketal,phosphoryl, sulphydryl, water-solubilizing groups, and alkyl groupsoptionally substituted by amino, C₁-C₄ alkyl-substituted amino,quaternary amino, carbonyl including aldehyde and ketone, acetal; ketal,halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate,carboxylate, amide, nitro, and groups reactive with amino, hydroxyl,aldehyde, phosphoryl, or sulphydryl groups;

[0023] A is selected from O, S and NR¹¹ where R¹¹ is the substitutedamino radical:

[0024] where R′ is selected from hydrogen, a C₁₋₄ alkyl and aryl and R″is selected from C₁₋₁₈ alkyl, aryl, heteroaryl, an acyl radical havingfrom 2-7 carbon atoms, and a thiocarbamoyl radical.

[0025] X and Y may be the same or different and are selected frombis-C₁-C₄ alkyl and C₄-C₅ spiro alkyl substituted carbon, oxygen,sulphur, selenium, CH═CH, and N—W wherein N is nitrogen and W isselected from hydrogen, a group —(CH₂)_(n)R¹² where n is an integer from1 to 26 and R¹² is selected from hydrogen, amino, aldehyde, acetal,ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate,carboxylate, substituted amino, quaternary amino, nitro, primary amide,substituted amide, and groups reactive with amino, hydroxyl, carbonyl,phosphoryl, and sulphydryl groups;

[0026] Z^(a) and Z^(b) each represent a bond or the atoms necessary tocomplete one, two fused or three fused aromatic rings each ring havingfive or six atoms, selected from carbon atoms and, optionally, no morethan two oxygen, nitrogen and sulphur atoms;

[0027] provided that when X and Y are other than carbon, at least one ofR¹-R⁹ comprises a reactive group for covalent reaction with a functionalgroup on a target material or comprises a functional group for covalentreaction with a reactive group on a target material, or,

[0028] when X and Y are different and are selected from O and Se, atleast one of R¹-R⁹ is other than hydrogen, methyl, phenyl or naphthyl.

[0029] Preferred R¹⁰ groups are selected from: hydrogen, halogen, amide,C₁-C₆ alkoxy, nitro, cyano, aryl, heteroaryl, sulphonate, quaternaryammonium, guanidinium, hydroxyl, phosphate, phosphonate, optionallysubstituted amino, azido, sulphydryl, carboxyl, carbonyl, reactivegroups, for example, succinimidyl ester, isothiocyanate, anhydride,haloacetamide, maleimide, sulphonyl halide, phosphoramidite, acidhalide, alkylimidate, hydrazide and carbodiimide; and groups reactivewith amino, hydroxyl, aldehyde, phosphoryl, or sulphydryl groups.

[0030] Preferably R¹ is selected from hydrogen, aryl, heteroaryl, cyano,halogen, alkyl groups of twenty-six carbon atoms or less and —(CH₂)_(n)Qwhere 1<n<26 and Q is selected from amino, aldehyde, sulphydryl,hydroxyl and groups reactive with amino, hydroxyl, aldehyde, phosphoryl,or sulphydryl groups and R², R³, R⁴ and R⁵ are hydrogen.

[0031] Suitably R¹² is selected from hydrogen, amino, sulphonate,carboxylate, aryl, hydroxyl, and groups reactive with amino, hydroxyl,carbonyl, phosphoryl, or sulphydryl groups.

[0032] Bis-substituted carbon includes bis C₁-C₄ alkyl groups and C₄-C₅spiro alkyl groups.

[0033] Alkyl is a straight or branched chain alkyl group containing from1-26 carbon atoms, suitably containing from 1-12 carbon atoms,preferably from 1-6 carbon atoms.

[0034] Aryl is an aromatic substituent containing one or two fusedaromatic rings containing 6-10 carbon atoms, for example phenyl ornaphthyl. The aryl may be optionally and independently substituted byone or more groups selected from groups —R¹⁰ and —L—R¹⁰ as hereinbeforedefined.

[0035] Heteroaryl is a mono- or bicyclic 5-10 membered aromatic ringsystem containing at least one and no more than 3 heteroatoms which maybe selected from N,O and S. The heteroaryl may be optionally andindependently substituted by one or more groups selected from groups—R¹⁰ and —L—R¹⁰ as hereinbefore defined.

[0036] Aralkyl is a C₁-C₅ alkyl group substituted by an aryl orheteroaryl group.

[0037] Halogen and halo-groups are those selected from fluorine,chlorine, bromine and iodine.

[0038] Specific examples of the groups R¹-R⁹ and R¹¹ and the groups withwhich those R-groups will react are provided in Table 1. In thealternative, the R¹-R⁹ and R¹¹ may be the functional groups of Table 1which would react with the reactive groups of a target molecule. TABLE 1Possible Reactive Substituents and Sites Reactive Therewith ReactiveGroups Corresponding Functional Groups succinimidyl esters primaryamino, secondary amino, hydroxyl anhydrides primary amino, secondaryamino, hydroxyl acyl azides primary amino. secondary aminosothiocyanates, isocyanates amino, thiol, hydroxyl sulphonyl chlorides,amino, hydroxyl sulphonyl fluorides substituted hydrazines, substitutedaldehydes, ketones hydroxylamines acid halides amino, hydroxylhaloacetamides, maleimides thiol, imidazoles, hydroxyl, aminocarbodiimides carboxyl groups phosphoramidite hydroxyl

[0039] In addition to those groups listed in Table 1, a number of othergroups are possible as reactive substituent in the R¹-R⁹ and R¹¹positions of the compounds of the present invention. For example, thereactive. groups which are especially useful for labelling targetcomponents with available amino and hydroxy functional groups include:

[0040] where n=0 or an integer from 1-10 and at least one of R¹³ or R¹⁴is a leaving group such as I, Br, or Cl.

[0041] Specific examples of possible R¹-R⁹ and R¹¹ groups that areespecially useful for labelling target components with availablesulphydryl functional groups include:

[0042] where n=0 or an integer from 1-10 and R¹⁵ is a leaving group suchas I or Br.

[0043] Specific examples of possible R¹-R⁹ and R¹¹ functional groupsthat are especially useful for labelling target components bylight-activated cross linking include:

[0044] For the purpose of increasing water solubility or reducingunwanted non-specific binding of the fluorescently-labelled component toinappropriate components in the sample or to reduce interactions betweentwo or more reactive chromophores on the labelled component which mightlead to quenching of fluorescence, the R¹-R⁹ and R¹¹ functional groupscan be selected from the well known polar and electrically chargedchemical groups. Examples of such groups are —E—F— where F is hydroxy,sulphonate, sulphate, carboxylate, substituted amino or quaternaryamino, and where E is a spacer group such as —(CH₂)_(n)— where n is 0-6.Useful examples of —E—F groups include C₁₋₆ alkyl sulphonates, such as—(CH₂)₃SO₃ ⁻ and —(CH₂)₄—SO₃ ⁻.

[0045] Exemplary compounds of the present invention which demonstratethe capability for adjusting fluorescence colour, water solubility, andthe position of the reactive or functional group are as follows:

[0046] i)6,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a;3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound I);

[0047] ii)8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium(Compound II);

[0048] iii)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III);

[0049] iv)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,glycinamide (Compound IV);

[0050] v)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-(2-aminoethylcarboxamide) (Compound V);

[0051] vi)6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VI);

[0052] vii)6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VII);

[0053] viii)6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzothiazolenine-indolenine-[3,2-a]-benzthiazolyl[3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VIII);

[0054] ix)6,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium(Compound IX);

[0055] x)6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenium-[3,2-a,3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound X).

[0056] The groups provided herein are not meant to be all-inclusive ofthose groups which can be incorporated at the R sites of the compoundsof the present invention. It will be understood that there are variousother groups which will react with groups on material that is to belabelled by the compounds of the present invention. Compounds producedby the incorporation of such other groups at the R¹-R⁹ and R¹¹ positionsare intended to be encompassed by the present invention.

[0057] The compounds of the present invention may be used in numerousbiological and non-biological applications. With respect tonon-biological applications, compounds of the present invention havingone or more uncharged groups at the R¹-R⁹ and R¹¹ positions, forexample, C₁₋₂₆ alkyl and aryl moieties may be dissolved in non-polarmaterials to provide fluorescent properties to those materials. Suchnon-polar materials include, for example, paints, polymers, waxes, oils,inks and hydrocarbon solvents. Another non-biological application of thepresent invention is to dissolve compounds of the present inventionhaving one or more charged and or polar groups at the R¹-R⁹ and R¹¹positions in polar solvents or other materials such as, for example,water, ethylene glycol, methyl alcohol, or a mixture of water and methylalcohol. Such charged R-groups include, for example, —NR₃ ⁻, —SO₃ ⁻,—PO₃ ⁻ and —COO⁻, while such polar R-groups include, for example,hydroxyl groups. With respect to biological applications, biologicalmolecules may be non-covalently labelled using the present complexes.For example, complexes of the present invention wherein at least one ofR¹-R⁹ and R¹¹ contains a charge, for example, quaternary amino, may beused to non-covalently bind to charged biological molecules such as, forexample, DNA and RNA. In addition, compounds of the present inventionwherein at least one of R¹-R⁹ and R¹¹ is an uncharged group, forexample, a long chain alkyl, may be used to bind to uncharged biologicalmolecules such as, for example, biological lipids.

[0058] Alternatively, the compounds of the present invention may containa polymerizable group suitable for the formation of a polymer containingthe complex. Suitable polymerizable groups are selected from acrylate,methacrylate, acrylamide, vinyl and styryl. Polymerization may becarried out with a suitably derivatized compound of this invention usedin conjunction with a second polymerizable monomer starting material,such as styrene or vinyltoluene, to form a copolymer containing thefluorescent compound. Alternatively the fluorescent compounds of theinvention need not have a polymerisable group, for example, the compoundmay be incorporated during polymerisation or particle formation or maybe absorbed into or onto polymer particles.

[0059] The dyes of the present invention can also be used as laser dyesaccording to the procedures set forth in U.S. Pat. No. 4,916,711 toBoyer and Morgan. Laser dyes must be fluorescent, must have a quantumyield greater than 0.56 or 0.57 and must be reasonably photostable. Thecompounds of the present invention satisfy each of these requirements.Further the dyes of the present invention can be used as textile dyes,photographic dyes and as organic conductors.

[0060] The compounds of the present invention may also be used tocovalently label a target material to impart fluorescent properties tothe target material. Covalent labelling using the compounds of thepresent invention may be utilized either in a biological or anon-biological application. Examples of target materials that may belabelled in non-biological applications include, for example,cellulose-based materials (including, for example, papers), textiles,petroleum-based products, photographic films, glasses, polymers and gelfiltration and chromatography media.

[0061] Covalent labelling using compounds of the present invention maybe accomplished with a target having at least one functional or reactivegroup as defined hereinbefore. The target may be incubated with anamount of a compound of the present invention having at least one ofR¹-R⁹ and R¹¹ that includes a reactive or functional group ashereinbefore defined that can covalently bind with the functional orreactive group of the target material. The target material and thecompound of the present invention are incubated under conditions and fora period of time sufficient to permit the target material to covalentlybond to the compound of the present invention.

[0062] R¹-R⁹ and R¹¹ can be chosen so that the compounds of the presentinvention react with different target compounds and, or to havedifferent spectral properties, thereby providing a number of relatedcompounds which can be used in multiplex analyses wherein the presenceand quantity of various compounds in a single sample must bedifferentiated based on the wavelengths and intensities of a number ofdetected fluorescence emissions.

[0063] The compounds of the present invention may be made soluble inaqueous, other polar, or non-polar media containing the material to belabelled by appropriate selection of R-groups.

[0064] The invention also relates to labelling methods wherein thecompounds of the present invention including at least one reactive groupat the R¹-R⁹ and R¹¹ positions covalently react with amino, hydroxyl,aldehyde, phosphoryl, carboxyl, sulphydryl or other reactive groups ontarget materials. Such target materials are include, but are not limitedto the group consisting of antibody, lipid, protein, peptide,carbohydrate, nucleotides which contain or are derivatized to containone or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl,phosphate and thiophosphate groups, and oxy or deoxy polynucleic acidswhich contain or are derivatized to contain one or more of an amino,sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphategroups, microbial materials, drugs, toxins, particles, plastics or glasssurfaces and polymers. Compounds of the present invention may also beused for coupling to additional fluorescent or non-fluorescent compoundsfor use in fluorescence resonance energy transfer complexes of the typedescribed in EPA 747700 or for fluorescence polarisation or fluorescencequenching-based applications.

[0065] In addition to the foregoing single-step labelling process, thepresent invention also relates to two-step labelling processes in which,in a first step, a compound of the present invention covalently reactswith and thereby labels a primary component, such as an antibody. In asecond or staining step of the two-step procedure, the fluorescentlylabelled primary component is then used as a probe for a secondarycomponent, such as an antigen for which the antibody is specific. Whenthe target of the so-labelled antibodies is a cell, the second step ofthe procedure may be used to determine the amount of labelled antibodieswhich are attached to that type of cell by determining the intensity ofthe fluorescence of the cells. By this two-step procedure, monoclonalantibodies and other components covalently labelled in the first stepwith the fluorescent compounds of the present invention could be used asantigen probes.

[0066] The compounds of the present invention can be used to determinethe concentration of a particular protein or other component in asystem. If the number of reactive groups on a protein which can reactwith a probe is known, the fluorescence per molecule can be known andthe concentration of these molecules in the system can be determined bythe total fluorescence intensity of the system. This particular methodcan be used to measure the concentration of various labelled analytesusing microtitre plate readers or other known immunofluorescencedetection systems.

[0067] The compounds of the present invention are also useful in assaymethodologies that employ fluorescent labels for the detection andmeasurement of analytes, using for example, fluorescence resonanceenergy transfer (FRET) based methods, fluorescence lifetime, or by meansof fluorescence polarization measurements.

[0068] The use of fluorescence resonance energy transfer dye pairs inbiological systems is well known and they have been used in thedetection of binding events or cleavage reactions in assays which employFRET. Examples of such assays include equilibrium binding assays, (eg.immunoassays, nucleic acid hybridisation assays, protein binding assaysand hormone receptor assays) and enzyme assays, such as proteolyticcleavage assays, the cleavage of a DNA or RNA molecule by a nuclease, ora lipid by a lipase.

[0069] Binding assays utilising compounds of the present invention maybe performed by binding one component of a specific binding pair with asecond component of the specific binding pair, the first component beinglabelled with a fluorescent donor dye according to the presentinvention, and the second component being labelled with a fluorescent(or quenching) acceptor dye, so as to bring about an energy transferrelationship between the first and second components, and detecting thebinding of the first and second components by measurement of the emittedfluorescence. Examples of specific binding pairs include, but are notrestricted to, antibodies/antigens, lectins/glycoproteins,biotin/(strept)avidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood that inthe present invention, any molecules which possess a specific bindingaffinity for each other may be employed, so that the dyes of the presentinvention may be used for labelling one component of a specific bindingpair, which in turn may be used in the detection of binding to the othercomponent.

[0070] The dyes of the present invention may also be used in an enzymecleavage assay format, in which the enzyme substrate, for example apeptide, comprises two components, one of which is labelled with afluorescent donor dye of the present invention, the second beinglabelled with a fluorescent (or quenching) acceptor dye and beingattached to the substrate in an energy transfer relationship on eitherside of the substrate bond to be cleaved. A known or a putative enzymeinhibitor compound may be optionally included in the reaction mixture.Cleavage of the substrate by the enzyme results in separation of thedonor and acceptor dyes, resulting in a loss of resonance energytransfer and a change in the fluorescence emission of the donor andacceptor species.

[0071] Suitable fluorescent acceptor dyes that can be combined with thedyes of the present invention to form energy transfer dye pairs includethe rhodamine and cyanine dyes. Particularly preferred are the cyaninedyes, including Cy5(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-dicarbocyanine),Cy5.5(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)-dibenzo-dicarbocyanine)and Cy7(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-tricarbocyanine).A suitable quenching acceptor dye is DABCYL(4-(4-dimethylaminophenyl)azobenzoic acid).

[0072] The dyes of the present invention may also be used in bindingassays or in enzyme cleavage assays, utilising fluorescence polarizationmeasurements. In a binding assay format, the assay of an analyte in asample may be performed by providing a specific binding partner for theanalyte, the specific binding partner being labelled with a dyeaccording to the present invention, measuring the fluorescencepolarization of the labelled specific binding partner, contacting theanalyte with the labelled specific binding partner under conditionssuitable for binding the analyte to form an analyte-specific bindingpartner complex and measuring the fluorescence polarization of thelabelled analyte-specific binding partner complex to determine theextent of binding.

[0073] In the second format, an assay for the detection of enzymeactivity may be configured as follows. A reaction mixture is prepared bycombining a protease enzyme and a fluorogenic substrate labelled with adye according to the present invention. A known or a putative inhibitorcompound may be optionally included in the reaction mixture. Cleavage ofthe substrate by the enzyme results in the production of labelledfragments. The progress of the reaction is monitored by observing thechange in fluorescence polarization.

[0074] The fluorescent compounds of the present invention can also beused in a detection method wherein a plurality of the fluorescentcompounds are covalently attached to a plurality of different primarycomponents, such as antibodies, each primary component being specificfor a different secondary component, such as an antigen, in order toidentify each of a plurality of secondary components in a mixture ofsecondary components. According to this method of use, each of theprimary components is separately labelled with a fluorescent compoundhaving a different light absorption and emission wavelengthcharacteristic compared with the dye molecules used for labelling theother primary components. The so-called primary components are thenadded to the preparation containing secondary components, such asantigens, and the primary components are allowed to attach to therespective secondary components for which they are selective.

[0075] Any unreacted probe materials may be removed from the preparationby, for example, washing, to prevent interference with the analysis. Thepreparation is then subjected to a range of excitation wavelengthsincluding the absorption wavelengths of particular fluorescentcompounds. A fluorescence microscope or other fluorescence detectionsystem, such as a flow cytometer or fluorescence spectrophotometer,having filters or monochrometers to select the rays of the excitationwavelength and to select the wavelengths of fluorescence is nextemployed to determined the intensity of the emission wavelengthscorresponding to the fluorescent compounds utilized, the intensity offluorescence indicating the quantity of the secondary component whichhas been bound with a particular labelled primary component. Knowntechniques for conducting multi-parameter fluorescence studies include,for example, multi-parameter flow cytometry.

[0076] In certain cases a single wavelength of excitation can be used toexcite fluorescence from two or more materials in a mixture where eachfluoresces at a different wavelength and the quantity of each labelledspecies can be measured by detecting its individual fluorescenceintensity at its respective emission wavelength. If desired, a lightabsorption method can also be employed.

[0077] The detection method of the present invention can be applied toany system in which the creation of a fluorescent primary component ispossible. For example, an appropriately reactive fluorescent compoundcan be conjugated to a DNA or RNA fragment and the resultant conjugatethen caused to bind to a complementary target strand of DNA or RNA.Appropriate fluorescence detection equipment can then be employed todetect the presence of bound fluorescent conjugates.

[0078] The present invention also relates to the covalent reactionbetween compounds of the present invention, and amine, hydroxy,aldehyde, sulphydryl, phosphoryl or other known functional groups onmaterials such as, for example, proteins, peptides, carbohydrates,nucleic acids, derivatized nucleic acids, lipids, certain otherbiological molecules, biological cells, soluble polymers, polymericparticles, polymer surfaces, polymer membranes, glass surfaces and otherparticles and surfaces. Because detecting fluorescence involves highlysensitive optical techniques, the presence of these dye “labels” can bedetected and quantitated even when the label is present in very lowamounts. Thus, the dye labelling reagents can be used to measure thequantity of a material that has been labelled.

[0079] Compared with, for example, the fluoresceins, the rigidizedtrimethine cyanines of the present invention are particularlyphotostable and are insensitive to pH changes between pH2 and pH10. Thecompounds of the present invention maximally absorb and emit light atwavelengths between 450 and 600 nm (green to orange region of thespectrum) and are therefore alternatives to Texas-Red, rhodamine,tetramethylrhodamine, X-rhodamine, BODIPY and fluorescein.

[0080] The present invention also provides a process for the preparationof a compound of formula (2) which comprises treating a compound offormula (A):

[0081] optionally substituted by groups R²-R⁹, wherein X, Y, Z^(a),Z^(b) and groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are as definedabove and R is methyl or ethyl, in mild acid solution, such as in aceticacid. Suitably the reaction mixture is heated under refluxingconditions, whereupon the rigidized carbocyanine dye precipitates fromsolution. Alternatively the reaction may be carried out in a strongermineral acid solution, such a sulphuric acid at lower temperatures, forexample ambient temperature. It may be advantageous to include in thereaction mixture, a solvent such as chloroform.

[0082] In the case of amino and hydrazino substituted carbocyanine dyesof the present invention, these may be prepared from intermediates ofgeneral structure (A) by including the appropriate amine or hydrazinoderivative in the acid solution used for preparing the rigidized dye.

[0083] Symmetrical compounds of structure (A) wherein X and Y are thesame and structures Z^(a) and Z^(b) are the same may be prepared byreacting a compound of structure (B):

[0084] optionally substituted with groups R², R³, R⁶ and R⁷ whereingroups, Z^(a) and X, R², R³, R⁶ and R⁷ are as hereinbefore defined and Ris methyl or ethyl, with an appropriate ortho ester such a ethylorthoformate in a suitable solvent medium to prepare the non-rigidizedtrimethine. The reaction is suitably carried out in solution in asolvent such as pyridine by heating under reflux. By suitablysubstituting the ortho ester, the central or meso carbon atom of theconjugated trimethine chain may be substituted with a variety ofsubstituents such as are represented by the group R¹. For example,replacement of the ethyl orthoformate in the reaction mixture with ethylorthoacetate will produce a trimethine cyanine dye in which the mesohydrogen is replaced with a methyl group.

[0085] Asymmetric compounds of structure (A) wherein X and Y aredifferent may be prepared by reacting a compound of formula (B),optionally substituted with groups R⁵ and R⁶ wherein groups R², R³, R⁵,R⁶, Z^(a) and X are as hereinbefore defined with a compound of structure(C):

[0086] optionally substituted by groups R⁸ and R⁹ wherein groups R¹, R⁴,R⁵, R⁸, R⁹, Y and Z^(b) are as hereinbefore defined, R is an alkyl groupsuch as methyl or ethyl, R^(a) is an acyl radical, such as acetyl,propionyl and benzoyl and R^(b) is hydrogen, an alkyl radical such asmethyl or ethyl, or an aryl radical such as phenyl. The reaction issuitably carried out in a 1:1 molar proportion in acetic anhydridesolution.

[0087] Intermediate compound (B) may be prepared by reacting thehydrohalide acid salt of the appropriate heterocyclic base of formula(D):

[0088] optionally substituted by groups R⁶ and R⁷, wherein X, Z^(a), R⁶and R⁷ are as defined above with a compound of formula (E):

[0089] wherein R² and R³ are as hereinbefore defined. The reaction isadvantageously carried out with reagent (E) in excess and in an inertsolvent of moderate polarity that dissolves both reagents, but which isnot a solvent for the reaction product. Examples of such media aresolvents such as acetonitrile. The reaction is suitably carried out atan elevated temperature, suitably 70° C. An acid such as acetic acid maybe added to the reaction mixture to facilitate the reaction. As aspecific example the hydrobromide salt of(2,3,3-trimethyl-3H-indol-5-yl)-acetic acid prepared by the method ofSouthwick et al (Org.Prep.Proceed.Int. 20, 279-84, 1989) is reacted withacrolein diethyl acetal in acetonitrile containing acetic acid assolvent. The reaction is suitably carried out at a temperature of 70° C.

[0090] Intermediates of formula (C) may be prepared by reaction of acompound of structure (B) containing a methyl substituent in the2-position with a formamidine of formula (F):

[0091] wherein R¹ and R^(b) are as hereinbefore defined and R^(c) isphenyl or substituted phenyl. Suitably the reaction is carried out bycondensing the quaternary salt of structure (B) with a 1.5 molar excessof the formamidine using an acid condensing agent, for example aceticanhydride, propionic anhydride, or glacial acetic acid. Acetic anhydrideis a particularly preferred condensing agent for the reaction.Alternatively, the condensation reaction may be performed without anyaddition; however acid condensation is to be preferred for theproduction of rigidized trimethine cyanine dyes substituted at thecentral or meso carbon atom of the trimethine chain. See, for example,British Patent No.412309.

[0092] Precursor compounds of formula such as (D) may be prepared bymethods well known to those skilled in the area. See for example U.S.Pat. No. 4,981,977, the entire disclosure of which is incorporated byreference.

[0093] It will be readily appreciated that certain compounds of formula(2) may be useful as intermediates for conversion to other compounds ofthe formula (2) by methods well known to those skilled in the art.Likewise, certain of the intermediates may be useful for the synthesisof derivatives of formula (2). The compounds of the present inventionmay be synthesized by the methods disclosed herein. Derivatives of thecompounds having a particular utility are prepared either by selectingappropriate precursors or by modifying the resultant compounds by knownmethods to include functional groups at a variety of positions. Asexamples, the complexes of the present invention may be modified toinclude certain reactive groups for preparing a fluorescent labellingreagent, or charged or polar groups may be added to enhance thesolubility of the compound in polar or nonpolar solvents or materials.As examples of conversions an ester may be converted to a carboxylicacid or may be converted to an amido derivative.

[0094] The following are specific examples of the synthesis of compoundsof the present invention and observed spectral data for those compounds.

EXAMPLE 1

[0095]6,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a;3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(R-Cy3.12.OH; Compound I)

[0096] 1.1 5-Carboxymethyl-2,3,3-trimethylindoline

[0097] 5-Carboxymethyl-2,3,3-trimethylindoline was prepared either bythe method of Southwick et al, Org.Prep.Proceed.Int., 20, 274-84,(1989), or alternatively as described below.

[0098] To a stirred solution of 4-aminophenylacetic acid (5 g, 33.1mmol) in a 3:2 water: conc. HCl (33 ml) solvent mixture at <0° C. wasadded dropwise a cooled (<0° C.) solution of sodium nitrite (2.7 g, 39mmol) in water (43 ml). The reaction mixture was then maintained at thereduced temperature for a further 30 minutes. A saturated aqueoussolution of sulphur dioxide (140 ml) was added and the reaction mixturewarmed to ambient temperature over 1 hour, then warmed for a furtherhour at 70° C. The reaction mixture was cooled rapidly and the solventremoved in vacuo. The yellow hydrazine intermediate product obtained wasredissolved in acetic acid (54 ml) and potassium acetate (7.05 g, 71.8mmol) and methyl-isopropyl ketone (6.84 g, 79.4 mmol) added at ambienttemperature. After 30 minutes the reaction mixture was warmed to 90° C.and stirred for a further 2 hours. The reaction mixture was cooled andthe reaction solvent removed in vacuo. The product was dissolved indichloromethane (100 ml) and washed with water (2×50 ml). The organicphase was dried over MgSO₄, filtered and concentrated in vacuo.5-ethoxycarbonyl-2,3,3-trimethylindolenine was obtained as a red solid(4.8 g, 67%). No purification was required; m/z (Maldi): 217.

[0099]1.21-(3,3-Diethoxypropyl-5-carboxymethyl-2,3,3-trimethylindolenine, ethylester

[0100] To a stirred solution of5-carboxymethyl-2,3,3-trimethylindolenine (2 g, 9.3 mmol) in ethanol (40ml) at ambient temperature was added hydrobromic acid (3.16 ml of 48%aqueous solution). After 1 hour the reaction solvent was removed invacuo. The hydrobromide salt was redissolved in acetonitrile (40 ml) andacetic acid (400 ml) and acrolein diethyl acetal (18.17 g, 140 mmol)added. The reaction mixture was warmed to 70° C. for 20 minutes. Thesolution was cooled and the reaction solvent removed in vacuo. Theproduct was purified by HPLC on a Rainin Dynamax C18, 8 μm column usinga 10-100% gradient elution of water/acetonitrile (containing 0.1% TFA)over 60 minutes at 20 ml/min. The product was obtained as a green oil(1.24 g, 36%); m/z (FAB⁺): 376.2.

[0101]1.35,5′-Dicarboxymethyl-1,1′-di-(3,3-diethoxypropyl)-indocarbocyanine-ethylester

[0102] To a stirred solution of1-(3,3-diethoxypropyl-5-carboxymethyl-2,3,3-trimethylindolenine, ethylester (356 mg, 0.95 mmol) in pyridine (10 ml) at 120° C. was addeddropwise, triethyl orthoformate (98 mg, 66 mmol) over 30 minutes. After2 hours the reaction mixture was cooled. The product was purified byHPLC on a Rainin Dynamax C18, 8 μm column using a 10-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 60 minutes at20 ml/min. The product was obtained as a pink solid (251 mg, 35%); λmax:555 nm; m/z (FAB⁺): 761.4.

[0103]1.46,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

[0104] To a stirred solution of5,5′-carboxymethyl-1,1-di-(3,3-diethoxypropyl)-indocarbocyanine, ethylester (100 mg, 0.132 mmol) in chloroform (10 ml) at ambient temperaturewas added 50% aqueous sulphuric acid (2 ml). After 30 minutes thereaction solvent was diluted with chloroform (10 ml) and washed withwater (3×10 ml). The organic phase was dried over NaSO₄, filtered andconcentrated in vacuo. The product was purified by HPLC on a RaininDynamax C18, 8 μm column using a 10-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a pink solid (65 mg, 90%); λmax 565 nm; m/z;m/z (FAB⁺): 539.2.

[0105]1.56,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-hydroxysuccinmidyl ester

[0106] To a mixture ofO-(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (5 mg, 0.012 mmol), and N,N′-diisopropylethylamine(4.08 mg, 0.032 mmol) in dimethylsulphoxide (500 μl) at ambienttemperature was added6,7,9,10-tetra-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(5 mg, 0.0089 mmol). The reaction mixture was stirred for 1 hr.Conversion to the N-hydroxysuccinimidyl ester derivative was confirmedby mass Spectroscopy and HPLC using a Phenomenex Jupiter C18 10 μmcolumn.

[0107] 1.6 Protein Labelling Procedure

[0108] A stock solution of the N-hydroxysuccinimidyl ester of Compound Iwas prepared in dry DMF (1 mg active ester/100 μl ). Sheep IgG (1 mg,6.45 mmol) was dissolved in 250 μl buffer solution (pH 9.4) and thedesired amount of dye was added during vigorous vortex mixing.Unconjugated dye was separated from the labelled protein by gelpermeation chromatography (0.7×20 cm column of Sephadex G-50) using pH 7buffer solution as eluant. Absorption spectra of the labelled antibodysolution was recorded (see FIG. 1). Dye to protein ratio for the samplewas determined using an equation below with measured values ofabsorbance of the labelled dye at 560 nm and the absorbance of proteinat 280 nm.$\frac{D}{P} = \frac{A_{dye} \times E_{prot}}{\left( {A_{280^{-}} \times A_{dye}} \right) \times E_{dye}}$

[0109] The factor X in the denominator accounts for the dye absorptionat 280 nm which is a % of the absorption of the dye at its maximumabsorption (A_(dye)). The value of X is 0.17 for a rigid dye.

[0110] 1.7 Comparison of Spectral Properties: Rigidized Cy3 (Compound I)and Open Chain Cy3

[0111] The spectral properties of the rigidized dye were compared withthe known open chain Cy3.18.OH and Cy3.10.OH dyes (FIG. 2). Theabsorption absorption and emission spectra are shown in FIGS. 3 and 4.The absorption maxima of the rigid dye shifted to the red by 12 nm inmethanol and as expected it is 10-12 time brighter that thenon-rigidized indocyanines. The results are shown in Table 2 below.TABLE 2 Dye in Methanol λ_(max) ε_(max) QY (φ) R-Cy3.12.OH (Compound I)565 584 0.8 Cy3.18.OH 555 570 0.09 Cy3.10.OH 555 578 0.08

[0112] Photo-bleaching of the dyes was studied under identicalconditions. Samples of equal concentrations of dyes (3 ml of 1.5×10⁻⁵mmol solution in water) were exposed to a laser line 514 nm (30 mV, 1 cmdiam. beam) and absorption was recorded in a few minutes of interval for50 minutes. The results are shown in FIG. 5. Rigidized dye is expectedto bleach faster. However, because of its high quantum yield, it isexpected that the rigidized dye will be suitable for flow cytometer andother imaging experiments where the sample is exposed for short timeperiods.

EXAMPLE 2

[0113]8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium(Compound II)

[0114] 2.1 6-Sulphonato-2,3,3-trimethyl-1H-benz[e]indolenine

[0115] A stirred solution of 2,3,3-trimethyl-1H-benz[e]-indolenine (100g, 478 mmol) in concentrated sulphuric acid (500 ml) was heated at 180°C. After 2 hours the solution was cooled to ambient temperature, thenpoured onto ice. The reaction mixture was made basic by adding 50%sodium hydroxide (3000 ml). The resulting precipitate was filtered,recrystallised from water and dried The product was obtained as a whitesolid (7.259, 54%).

[0116] 2.21-(3,3-Diethoxypropyl)-6-sulphonato-2,3,3-trimethyl-1H-benz[e]indolenine

[0117]1-(3,3-Diethoxypropyl)-6-sulphonato-2,3,3-trimethyl-1H-benz[e]indoleninewas prepared by reaction of6-sulphonato-2,3,3-trimethyl-1H-benz[e]indolenine (25 mg, 0.0087 mmol)with acrolein diethyl acetal (169 mg, 1.3 mmol) and acetic acid (10 μl)in acetonitrile (2 ml) by an analogous method to that described inSection 1.2. The compound was not purified, as decomposition wasobserved. The product was obtained as a pale yellow oil.

[0118] 2.36,6′-Disulphonato-1,1′-di-(3,3-diethoxypropyl)-benz[e]indo-carbocyanine

[0119]6,6′-Disulphonato-1,1-di-(3,3-diethoxypropyl)-benz[e]indocarbocyaninewas prepared by reaction of1-(3,3-diethoxypropyl)-6-sulphonato-2,3,3-trimethyl-1H-benz[e]indoleninewith triethyl orthoformate (51.2 mg, 0.035 mmol) in pyridine (5 ml) byan analogous method to that described in Section 1.3. The compound waspurified by HPLC on a Phenomenex Jupiter C18, 10 μm column using 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 30minutes at 4 ml/min. The product was obtained as a pink/purple solid;λmax (MeOH) 580 nm, m/z (Maldi): 852.

[0120] 2.48,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium

[0121]8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-iumwas prepared by reaction of6,6′-disulphonato-1,1-di-(3,3-diethoxypropyl)-benz[e]indocarbocyanine (3mg, 0.0035 mmol) in chloroform (5 ml) and 50% sulphuric acid (1 ml) byan analogous method to that described in section 1.4.

[0122] The compound was purified by HPLC on a Phenomenex Jupiter C18, 10μm column using 0-100% gradient elution of water/acetonitrile(containing 0.1% TFA) over 30 minutes at 4 ml/min. The product wasobtained as a luminescent pink/purple solid; λmax (MeOH) 598 nm; m/z(Maldi): 684.

EXAMPLE 3

[0123]6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III)

[0124] 3.1 5-Sulphonato-2,3,3-trimethylindolenine

[0125] To a stirred solution of 4-hydrazinobenzene sulphonic acid (68 g,361 mmol) in acetic acid (205 ml) at ambient temperature was added3-methyl-2-butanone (88.44 g, 1027 mmol). The reaction was heated underreflux. After 3 hours the solution was cooled and the resulting pinkprecipitate was filtered, washed with acetic acid (50 ml) and dried. Theproduct was redissolved in methanol (800 ml) and a solution of potassiumhydroxide (20.4 g, 364 mmol) in isopropanol (200 ml) was added. Theyellow solid obtained was filtered and dried (48 g, 56%); m/z (FAB⁺):240.

[0126] 3.2 1-(3,3-Diethoxypropyl)-5-sulphonato-2,3,3-trimethylindolenine

[0127] 1-Diethoxy propyl-5-sulphonato-2,3,3-trimethylindolenine wasprepared by reaction of 5-sulphonato-2,3,3-trimethylindolenine potassiumsalt (1 g, 3.88 mmol) with acrolein diethyl acetal (8.54 g, 65.6 mmol)and acetic acid (1 ml) in acetonitrile (40 ml) using an analogous methodto that described in Section 1.2. The compound was purified by HPLC on aRainin Dynamax C18, 8 μm column using 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a green oil (740 mg, 52%); m/z: (FAB⁺)370.1.

[0128] 3.35-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine

[0129] To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2,3,3-trimethylindolenine-ethylester (1.16 g, 3.09 mmol) in acetic anhydride (25 ml) was addedN,N′-diphenylformamidine (908 mg, 4.63 mmol). The reaction mixture waswarmed to 110° C. After 30 minutes the solution was cooled and thereaction solvent removed in vacuo. The product was purified by HPLC on aRainin Dynamax C18, 8 μm column using 10-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a pale brown oil (634 mg, 40%); m/z (FAB⁺):521.2.

[0130] 3.45-Carboxymethyl-5′sulphonato-1,1′-di-(3,3-diethoxyoropyl)-indocarbocyanine,ethyl ester

[0131] To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine(96 mg, 0.19 mmol ) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride(5 ml) at ambient temperature was added a solution of5-sulphonato-1-(3,3-diethoxypropyl)-2,3,3-trimethylindolenine (67.7 mg,0.19 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5 ml).The reaction mixture was warmed to 70° C. for 5 hours. The solution wascooled and the reaction solvent removed in vacuo. The product waspurified by HPLC on a Rainin Dynamax C18, 8 μm column using a 10-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pink solid (33 mg,24%); λ max (MeOH) 555 nm; m/z (FAB⁺): 755.3.

[0132] 3.56,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;56-c′]dipyridin-5-ium

[0133]6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of5-carboxymethyl-5'sulphonato-1-di-(3,3-diethoxypropyl)-indocarbocyanine,ethyl ester (33 mg, 0.044 mmol) in chloroform (10 ml) and 50% sulphuricacid (2 ml) by an analogous method to that described in Section 1.4. Theproduct was purified by HPLC on a Rainin Dynamax C18 column using a0-100% gradient elution of water/acetonitrile (containing 0.1% TFA) over60 minutes at 20 ml/min. The product was obtained as a pink solid (18.7mg, 76%); λ max (MeOH) 563 nm; m/z (FAB⁺): 561.

[0134] 3.66,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-hydroxysuccinimidyl ester

[0135] To a mixture ofO-(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (5 mg, 0.012 mmol), and N,N′-diisopropylethylamine(4.08 mg, 0.032 mmol) in dimethyl sulphoxide (500 μl) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(5mg, 0.0089 mmol). The reaction was stirred for 1 hr. Conversion to theN-hydroxysuccinimidyl ester derivative was confirmed by massspectroscopy and HPLC using a Phenomenex Jupiter C₁₈ ₁₀ μm column.

EXAMPLE 4

[0136]6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,glycinamide (Compound IV)

[0137] To a mixture ofO-(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (1 mg, 0.0024 mmol) and N,N′-diisopropylethylamine(0.82 mg, 0.0032 mmol) in dimethylformamide (100 ml) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(1 mg, 0.0018 mmol). After 1 hour, glycine (0.2 mg, 0.0027 mmol) wasadded and the solution stirred for a further 3 hours. The product waspurified by HPLC on a Phenomenex Jupiter C_(18, 10) μm column, using0-100% gradient elution of water/acetonitrile (containing 0.1% TFA) at 4ml/min. The product was obtained as a pink solid (0.22 mg, 30%); m/z(Maldi): 618.

EXAMPLE 5

[0138]6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-(2-aminoethylcarboxamide) (Compound V)

[0139] To a mixture ofO-(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (5 mg, 0.012 mmol) and N,N′-diisopropylethylamine(4.08 mg, 0.032 mmol) in dimethylformamide (500 ml) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(5 mg, 0.0089 mmol). After 1 hour tert-butyl-N-(2-aminoethyl)-carbamate(1.4 mg, 0.0089 mmol) was added and the solution stirred for a further 2hours. The solvent was removed in vacuo. The product was dissolved in a95% aqueous trifluoroacetic acid solution and stirred for 2 hours. Theproduct was purified by HPLC on a Phenomenex Jupiter C18, 10 μm column,using gradient elution of acetonitrile/water (containing 0.1% TFA). Theproduct was obtained as a pink solid (1.6 mg, 30%); m/z (FAB⁺): 603.1.

EXAMPLE 6

[0140]6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18.18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VI)

[0141] 6.1 5-Phthalimidomethyl-2,3,3-trimethylindolenine

[0142] To a stirred solution of 2,3,3,-trimethylindolenine (20 g, 126mmol) in concentrated sulphuric acid (100 ml) at ambient temperature wasadded portionwise N-hydroxymethylphthalimide (20 g, 114 mmol). After 70hours the reaction mixture was poured onto ice and made basic withconcentrated ammonium hydroxide. The resulting precipitate was filteredand dried. The product was obtained as a yellow solid (34.84 g, 87%).

[0143] 6.2 5-Aminomethyl-2,3,3-trimethylindolenine

[0144] To a stirred solution of5-phthalimidomethyl-2,3,3-trimethylindolenine (10 g, 31.4 mmol) inmethanol (50 ml) at ambient temperature was added hydrazine hydrate(13.1 g 409 mmol). After 20 hours a precipitate was formed. The reactionmixture was adjusted to pH 1 with 6N HCl and the solvent removed invacuo. The solid obtained was suspended in 1N HCl and filtered throughcelite. The filtrate was washed with dichloromethane (3×40 ml) and theaqueous phase adjusted to pH 12 with 6N NaOH, then extracted withdichloromethane (3×40 ml). The organic phase was dried over Na₂SO₄,filtered and concentrated in vacuo to give a pale yellow solid (4.98 g,84%); m/z (Maldi): 188.

[0145] 6.3 5-(N-Formyl)aminomethyl-2,3,3-trimethylindolenine

[0146] A stirred solution of 5-aminomethyl-2,3,3-trimethylindolenine(4.98 g, 26.5 mmol) in methyl formate (30 ml) was refluxed under anitrogen atmosphere for 22 hours. The solution was cooled and thesolvent removed in vacuo. The product was obtained as pale brown oil(5.4 g, 94%); m/z (FAB⁺): 217.1.

[0147] 6.41-(3,3-Diethoxypropyl)-5-(N-formylaminomethyl-2,3,3-trimethylindolenine

[0148]1-(3,3′-Diethoxypropyl)-5-(N-formyl)aminomethyl-2,3,3-trimethylindoleninewas prepared by reaction of5-(N-formyl)aminomethyl-2,3,3-trimethylindolenine (1 mg, 24.8 mmol) withacrolein diethyl acetal (9 g, 69.1 mmol) and acetic acid (1 ml) inacetonitrile (40 ml) by an analogous method to that described in Section1.2. The product was purified by HPLC no on a Rainin Dynamax C18, 8 μmcolumn using a 0-100% gradient elution of water/acetonitrile (containing0.1% TFA) over 60 minutes at 20 ml/min The product was obtained as ayellow oil (1.10 mg, 69%); m/z (Maldi): 347.

[0149] 6.51-(3,3-Diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-5-sulphonato-2,3,3-trimethylindolenine

[0150]1-(3,3-Diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-5-sulphonato-2,3,3-trimethylindoleninewas prepared by reaction of5-sulphonato-1-(3,3′-diethoxypropyl)-2,3,3-trimethylindolenine (100 mg,0.27 mmol) [prepared as described in Section 3.2] withN,N′-diphenylformamidine (79 mg, 0.405 mmol) in acetic anhydride (20 ml)by an analogous method to that described in Section 3.3. The product wasnot purified, as decomposition was observed. The product was obtained asa yellow oil.

[0151] 6.61,1-Di-(3,3′-diethoxypropyl)-5-(N-formyl)aminomethyl-5′-sulphonato-indocarbocyanine.

[0152] To a stirred solution of5-sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine(37mg, 0.072 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5ml) at ambient temperature was added a solution of1-(3,3-diethoxypropyl)-5(N-formyl)aminomethyl-2,3,3-trimethylindolenine(25 mg, 0.072 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride(5 ml). The reaction mixture was warmed to 70° C. for 5 hours. Thesolution was cooled and the solvent removed in vacuo. The product waspurified by HPLC on a Rainin dynamax C18, 8 μm column using a 10-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pink solid (16 mg,15%); λ max (MeOH); 555 nm, m/z (FAB⁺): 726.1.

[0153] 6.76,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

[0154]6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of1,1-Di-(3,3-diethoxypropyl)-5-(N-formyl)aminomethyl-5′-sulphonato-indocarbocyanine(5 mg, 0.0069 mmol) in chloroform (5 ml) and 50% sulphuric acid (1 ml)by an analogous method to that described in Section 1.4. The product waspurified by HPLC on a Rainin Dynamax C18, 8 μm column using a 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pink solid (3.3 mg,90%); λ max (MeOH) 564 nm; m/z (Maldi): 560.

[0155] 6.86,7,9,10-Tetrahydro-2-aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

[0156] A solution of6,7,9,10-tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(2 mg) in conc. HCl: methanol [1:12] (5 ml) was stirred for 12 hours.The reaction solvent was removed in vacuo and the product purified byHPLC on a Phenomenex Jupiter C18, 10 μm column using 0-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4ml/min. The product was obtained as a pink solid (1.9 mg, 50%); λ max(MeOH) 560 nm; m/z (Maldi): 532.

EXAMPLE 7

[0157]6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VII)

[0158] 7.1 5-Hydroxyethyl-2,3,3-trimethylindolenine

[0159] To a stirred solution of 4-(2-hydroxy)ethyl-aniline (10 g, 73mmol) in a 3:2 water: conc. HCl (73 ml) solvent mixture at <0° C. wasadded dropwise a cooled (<0° C.) solution of sodium nitrite (6 g, 73mmol) in water (86 ml). The reaction mixture was then maintained at thereduced temperature for a further 30 minutes. A saturated solution ofsulphur dioxide (150 ml) was added and the reaction warmed to ambienttemperature over 1 hour, then warmed for a further hour at 70° C. Thereaction mixture was cooled rapidly and the solvent removed in vacuo.The yellow hydrazino intermediate product obtained was redissolved inacetic acid (120 ml) and potassium acetate (16 g, 163 mmol), andmethyl-isopropyl ketone (15.5 g, 180 mmol) added at ambient temperature.After 30 minutes the reaction mixture was warmed to 90° C. and stirredfor a further 2 hours. The reaction mixture was cooled and the solventremoved in vacuo. The product was dissolved in dichloromethane (100 ml)and washed with water (2×50 ml). The organic phase was dried over MgSO₄,filtered and concentrated in vacuo. The product was purified by HPLC ona Rainin Dynamax C18, 8 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a yellow oil (1.18 g, 8%; m/z (FAB⁺): 204.1.

[0160] 7.21-(3,3-Diethoxypropyl)-5-hydroxyethyl-2,3,3-trimethylindolenine

[0161] 1-(3,3-Diethoxypropyl)-5-hydroxyethyl-2,3,3-trimethylindoleninewas prepared by reaction of 5-hydroxyethyl-2,3,3-trimethylindolenine(118 mg, 0.072 mmol) with acrolein diethyl acetal (1.13 g, 8.68 mmol),acetic acid (100 μl) in acetonitrile (4 ml) by an analogous method tothat described in Section 1.2. The product was purified by HPLC on aPhenomenex Jupiter C18, 10 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 30 minutes at 4 ml/min.The product was obtained as a pale brown oil (24 mg, 12%); m/z (Maldi):331.

[0162] 7.3 5-Sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine

[0163]5-Sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindoleninewas prepared by reaction of5-sulphonato-1-(3,3′diethoxypropyl)-2,3,3-trimethylindplenine (100 mg,0.27 mmol) [prepared as described in Section 3.2] withN,N′-diphenylformamidine (79 mg, 0.405 mmol) in acetic anhydride (20 ml)by an analogous method to that described in Section 3.3. The product wasnot purified, as decomposition was observed. The product was obtained asa yellow oil.

[0164] 7.4 1-(3,3-Diethoxyoropyl)-5-hydroxyethyl-indocarbocyanine

[0165] To a stirred solution of5-sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine(30 mg, 0.06 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5ml) at ambient temperature was added a solution of1-(3,3′-diethoxypropyl)-5-hydroxyethyl-2,3,3-trimethylindolenine (20 mg,00.6 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5 ml).The reaction mixture was warmed to 70° C. for 5 hours. The solution wascooled and the reaction solvent removed in vacuo. The product waspurified by HPLC on a Phenomenex Jupiter C18, 10 μm column using a0-100% gradient elution of water/acetonitrile (containing 0.1% TFA) over30 minutes at 4 ml/min. The product was obtained as a pink solid (8.6mg, 10%), λ max (MeOH) 555 nm; m/z (Maldi):712.

[0166] 7.56,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

[0167]6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of1,1-di-(3,3′diethoxypropyl)-5-hydroxyethyl-indocarbocyanine (4.3 mg,0.06 mmol) in chloroform (5 ml) and 50% sulphuric (1 ml) according tothe method described in Section 1.4. The product was purified by HPLC ona Phenomenex Jupiter C18, 10 μm column using a 0-100% gradient elutionof water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4 ml/min.The product was obtained as a pink solid (2.8 mg,90%), λ max 565 nm; m/z(Maldi): 547.

EXAMPLE 8

[0168]6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzothiazolenine-indolenine-[3,2-a]-benzthiazolyl[3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VIII)

[0169] 8.1 1-(3,3-Diethoxypropyl)-2-methylbenzothiazole

[0170] 1-(3,3-Diethoxypropyl)-2-methyl-benzothiazole was prepared byreaction of 2-methylbenzothiazole (125 mg, 0.84 mmol) with acroleindiethyl acetal (1.64 g, 12.6 mmol) and acetic acid (100 μl) inacetonitrile (4 ml) by a method analogous to that described in Section1.2. The product was purified by HPLC on a Rainin Dynamax C18, 8 μmusing a 0-100% gradient elution of water/acetonitrile (containing 0.1%TFA) over 60 minutes at 20 ml/min. The product was obtained as acolourless oil (220 mg, 95%); m/z (FAB⁺): 280.

[0171] 8.25-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine,ethyl ester

[0172]5-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine,ethyl ester was prepared by reaction of5-carboxymethyl-1-(3,3-diethoxypropyl)-2,3,3-trimethylindoline, ethylester [prepared as described in Section 1.2] (1.16 g, 3.09 mmol) withN,N′-diphenylformamidine (908 mg, 4.63 mmol) in acetic anhydride (25 ml)by a method analogous to that described in Section 3.3. The product waspurified by HPLC on a Rainin Dynamax C18, 8 μm column using 10-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pale brown oil (634mg, 40%). m/z (FAB⁺): 521.

[0173] 8.314-Carboxymethyl-1,1′-di(diethoxyoropyl)-benzthiazolenine-indocarbocyanine,ethyl ester

[0174] To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine,ethyl ester (28 mg, 0.054 mmol ) in 4.5:4.5:1 pyridine:aceticacid:acetic anhydride (5 ml) at ambient temperature was added a solutionof 1-(3,3-diethoxypropyl)-2-methyl-benzothiazole (15.1 mg, 0.054 mmol)in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5 ml). The reactionmixture was warmed to 70° C. for 5 hours. The solution was cooled andthe reaction solvent removed in vacuo. The product was purified by HPLCon a Phenomenex Jupiter C18, 10 μm column using a 0-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4ml/min. The product was obtained as a pink solid (14.3 mg, 20%), λ max549 nm; m/z (FAB⁺): 665.3.

[0175] 8.46,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzathiozolenine-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

[0176]6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzathiozolenine-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of (name)(5 mg, 0.0075 mmol) in chloroform (5ml) and 50% sulphuric acid (1 ml) by an analogous method to thatdescribed in section 1.4. The product was purified by HPLC on aPhenomenex Jupiter C18, 10 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 30 minutes at 4 ml/min.The product was obtained as a pink solid (3.2 mg, 90%); λ max 562 nm;m/z (Maldi): 471.

EXAMPLE 9

[0177]6,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium(Compound IX)

[0178] 9.11,1-Di-(3,3-diethoxypropyl)-5,5′-disulphonato-indocarbocyanine.

[0179] 1,1-Di-(3,3-diethoxypropyl)-5,5′-disulphonato-indolcarbocyaninewas prepared by the reaction of1-diethoxypropyl-5-sulphonato-2,3,3-trimethylindolenine (25.4 mg, 0.069mmol) [prepared as described in Section 3.2] with triethyl orthoformate(40.7 mg, 0.275 mmol) in pyridine (5 ml) by an analogous method to thatdescribed in Section 1.3. The product was purified by HPLC on a RaininDynamax C18, 8 μm column using a 0-100% gradient elution ofwater/acetonitrile over 60 minutes at 20 ml/min. The product wasobtained as a pink solid (6.3 mg, 12%); λ max (MeOH) 555 nm; m/z(Maldi):750.

[0180] 9.26,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium

[0181] To a stirred solution of1,1-di-(3,3-diethoxypropyl)-5,5′-disulphonato-indolcarbocyanine (2 mg,0.0027 mmol) in anhydrous acetic acid (2 ml) was added 4-hydrazinophenylacetic acid (0.89 mg, 0.0054 mmol) and reaction was warmed to 100° C.After 10 minutes the solution was cooled and the reaction solventremoved in vacuo. The product was purified by HPLC on a PhenomenexJupiter C18, 10 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 30 minutes at 8 ml/min.The product was obtained as two diastereomeric compounds, both werepink/purple solids (0.04 mg, 2%, 0.06 mg, 3%). λ max (MeOH) 563 nm; m/z(FAB⁺): 717.21, (FAB⁺): 717.20.

EXAMPLE 10

[0182]6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenium-[3,2-a,3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound X)

[0183] 10.1 1-[2-(1,3-Dioxalan-2-yl)ethyl]-2-methyl-quinoline bromide

[0184] 2-Methyl quinoline (1.6 g, 0.011 mol) and2-(2-bromoethyl)-1,3-dioxolane (7.7 g, 0.043 mol) were heated togetherat 85° C. for 16 hrs. On cooling the reaction mixture was diluted withdiethyl ether and the resultant solid filtered off, washed with etherand dried. 1-[2-(1,3-dioxalan-2-yl)ethyl]-2-methyl-quinoline bromide wasobtained as a brown solid (0.98 g, 27%). m/z (FAB⁺) 244.

[0185] 10.25-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenineethyl ester

[0186]5-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenineethyl ester was prepared by reaction of5-carboxymethyl-1-(3,3-diethoxypropyl)-2,3,3-trimethylindoline ethylester [prepared as described in section 1.2] (1.16 g, 3.09 mmol) withN,N′-diphenylformamidine (908 mg, 4.63 mmol) in acetic anhydride (25 ml)by a method analogous to that described in Section 3.3. The product waspurified by HPLC on a Rainin Dynamax C18 column using 10-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 60 minutes at20 ml/min. The product was obtained as a pale brown oil (634 mg, 40%).m/z (FAB⁺): 521

[0187] 10.314-Carboxymethyl-1-[2-(1,3-dioxalan-2-yl)ethyl]-1′-(3,3-diethoxypropyl)-quinolino-indocarbocyanine,ethyl ester.

[0188] To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenineethyl ester (16.1 mg, 0.031 mmol ) in ethanol (0.5 ml) at ambienttemperature was added a solution of1-[2-(1,3-dioxalan-2-yl)ethyl]-2-methyl-quinoline-bromide (10 mg, 0.031mmol) in ethanol (0.5 ml) and triethylamine (125 μl, 0.9 mmol). After 1hour the reaction solvent was removed in vacuo and the product purifiedby HPLC on a Phenomenex Jupiter C18, 10 mm column using a 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 30minutes at 4 ml/min. The product was obtained as a purple solid (10 mg,51%), λ max 567 nm; m/z (FAB⁺): 629.

[0189] 10.46,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-guinolino-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

[0190]6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinoline-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of(14-carboxymethyl-1-[2-(1,3-dioxalan-2-yl)ethyl]-1′-(3,3-diethoxypropyl)-quinolino-indocarbocyanine,ethyl ester (5 mg, 0.0079 mmol) in chloroform (2 ml) and 50% sulphuricacid (0.4 ml) by an analogous method to that described in section 1.4.The product was purified by HPLC on a Phenomenex Jupiter C18, 10 mmcolumn using a 0-100% gradient elution of water/acetonitrile (containing0.1% TFA) over 30 minutes at 4 ml/min. The product was obtained as apurple solid (1.6 mg, 44%); λ max 584 nm; m/z (FAB⁺):465.

EXAMPLE 11

[0191] Preparation of Rigid Cy-3-Cy-5 Conjugate (Compound (X)

[0192] To a mixture of0-(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (1 mg, 0.0024 mmol), and N,N′-diisopropylethylamine(0.68 mg, 0.007 mmol) in dimethyl sulphoxide (100 μl) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-6,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(1 mg, 0.0018 mmol). After 1 hour diisopropylethylamine (0.23 mg, 0.0018mmol) and a solution of5-aminomethyl-5′-sulphonato-1-methyl-1′-ethylindodicarbocyanine (0.9 mg,0.0018 mmol) in dimethyl sulphoxide (100 μl) was added at ambienttemperature. After a further 48 hours the product was purified by HPLCon a Phenomenex Jupiter C18, 10 μm column using a 0-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4ml/min. The product was obtained as a blue solid; λ_(abs) (MeOH) 561 nmand λ_(em) 647 nm; m/z (FAB⁺): 1050.

EXAMPLE 12

[0193] Protein:Peptide Polarization Binding Assay

[0194] 12.1 Synthesis of Labelled Peptide Liqand

[0195] A peptide of sequence E-pY-I-N-Q-S-V-P-K (E9K) was prepared bysolid phase synthesis on an Applied Biosystems 431 A peptide synthesizerusing standard methods and materials. An excess of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III), N-hydroxysuccinimide ester was coupled in DMSO in thepresence of diisopropylethylamine to the free N-terminus of theprotected peptide whilst still attached to the solid phase. Afterdeprotection for two hours, the crude labelled peptide was purified byreverse phase HPLC, using a gradient from water/0.1% TFA towater:acetonitrile (40:60)/0.1% TFA over 60 minutes.

[0196] 12.2 Binding Assay

[0197] Various concentrations of Grb2 glutathione-S-transferase fusionprotein and E9K labelled with Compound III in 20 mM MOPS pH7.4/10 mMDTT/005% Tween 20 were incubated in a final volume of 150 μl in black96-well microplates (Dynatech) for 60 minutes. Non-specific binding wasdefined using 100 μM unlabelled peptide. Polarization values were readon a Fluorolite FPM2™ plate reader (Jolley Research and Consulting Inc.)using a 530DF30 filter for excitation and 590DF45 filter for emission.The results are shown in FIG. 6 and indicate the specific binding (asdetermined by change in polarization) of the Compound III-labelledpeptide with the Grb2 protein.

EXAMPLE 13

[0198] Nucleic Acid FRET Hybridization Assay

[0199] 13.1 Probe Preparations

[0200] Unlabelled target oligonucleotide (5′TAC CCA GAC GAG CAA-biotin3′) and complementary unlabelled probe oligonucleotide (5′ TTG CTC GTCTGG GTA 3′) were synthesised on an Applied Biosystems 391 DNAsynthesiser using standard methods and materials. The oligonucleotideswere deprotected for 17 hours at 40° C. and purified by reverse phaseHPLC using a C18 column and a 40% TEAA/acetonitrile gradient. Thedesired peaks were collected, freeze dried and the samples wereresuspended in sterile H₂O.

[0201] A second set of target and probe oligonucleotides weresynthesised as described, but an amino group was added to the 5′terminal (5′ C₇ amino-modifier TAC CCA GAC GAG CAA-biotin 3′ and 5′ C₇amino modifier TTG is CTC GTC TGG GTA 3′).

[0202] Amino modified target and probe oligonucleotides were incubatedwith a 10-fold molar excess of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III), N-hydroxysuccinimide ester and Cy5 NHS-ester dye(Amersham Pharmacia Biotech) respectively, in 0.1 M sodium bicarbonatebuffer (pH9), overnight at 22° C. The following morning, theoligonucleotides were ethanol precipitated and the resulting pelletswere resuspended in H₂O. Labelled oligonucleotides were purified byreverse phase HPLC using a C18 column and a 60% TEAA/acetonitrilegradient and the desired peaks collected and freeze-dried. Residues wereresuspended into H₂O and concentration of recovered material wasdetermined.

[0203] 13.2 Binding Assay

[0204] Wells of a black, streptavidin coated 96-well plate were coatedwith either unlabelled or Compound III-labelled target oligonucleotides(20 pmol/well diluted in 100 μl PBS/1MgCl₂) for 120 minutes at ambienttemperature. Any unbound material was removed by washing wellsvigorously with assay buffer (PBS/1 mM MgCl₂/0.1% BSA). Unlabelled orCy5-labelled probe oligonucleotides were diluted to 0.2 pmol/μl assaybuffer, and 100 μl was incubated with coated wells at ambienttemperatures for 120 minutes to allow probe hybridisation. Finally,wells were washed vigorously with PBS and fluorescence intensity wasmeasured on a fluorescence plate reader using a 560 nm excitation filterand a 670 nm emission filter (FIG. 7).

[0205] Wells coated with unlabelled target oligonucleotide and incubatedwith either unlabelled or Cy5-labelled probe gave residual backgroundfluorescence signals. Similarly, wells coated with Compound III-labelledtarget oligonucleotide and incubated with unlabelled probe gave lowfluorescence signals. Wells coated with Compound III-labelled targetoligonucleotide and incubated with Cy5-labelled probe gave a strongfluorescence signal demonstrating that FRET can occur between CompoundIII and Cy5.

EXAMPLE 14

[0206] Protein:DNA Direct Intensity Binding Assay

[0207] 14.1 Preparation of Reagents

[0208] All HPLC purified oligonucleotides were obtained from GenosysBiotechnologies Ltd. Equimolar amounts of a biotinylated coding strand(5′ Biotin-GATCTAGGGACTTT CCGCG 3′) and an unmodified non-coding strand(5′0 ATCCCTGAAAGGCGCCTA 3′) specific for NF-kB were incubated togetherin a boiling water bath for 3 minutes and allowed to anneal by coolingover 2 hours.

[0209] Anti-GST antibody (3 mg/ml, 1.5 mg supplied/vial (0.5 ml) fromMolecular Probes) was dialysed against 1 litre of 0.15M sodium chloridefor 4 hours at room temperature and dialysis was continued overnight at4° C. in a fresh solution of 0.15M sodium chloride. The followingmorning the antibody was dialysed against 1 litre of 0.1M sodiumhydrogen carbonate for a maximum of 4 hours.

[0210] A 1 mg/ml solution of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III), N-hydroxysuccinimide ester in DMSO was added graduallywith stirring to the antibody at a ratio of 0.10 mg dye:0.33 mgantibody. The solution was mixed for a further 45 minutes at roomtemperature in the dark. Free dye was removed by dialysis against 1litre of 0.15M sodium chloride for 4 hours at room temperature andovernight at 40° C. against 1 litre of fresh 0.15M sodium chloride.Finally, the antibody was dialysed against 1 litre of 0.01M PBS/0.01%sodium azide for 4 hours at room temperature and then overnight at 4° C.against 1 litre of 0.01M PBS/0.01% sodium azide. [All dialyses wereperformed in the dark following labelling.]

[0211] 14.2 Binding Assay

[0212] Biotin-labelled NF-kB-specific dsDNA (2.5 pmol, diluted in0.01MMgCl₂) was added to each well (final volume, 100 μl) of a 96-wellstreptavidin coated microplate (Boehringer Mannheim) and incubated atroom temperature for 2 hours. Following washing with 0.01M phosphatebuffer (pH7.5) containing 0.05% Tween 20, 5 pmol/well of p65GST wasadded in 10 mM Hepes, 0.2 mM sodium acetate, 0.05% NP40, 1 mg/ml BSA and5 mM DTT (blanks contained no p65GST), in the presence or absence ofeither 200 pmol/well p65 (specific competitor) or casein (non-specificcompetitor). Both proteins were diluted in the Hepes buffer as above;final well volume was 100 μl. The plate was agitated at room temperaturefor 30 minutes and left to stand for a further 30 minutes. Followingwashing in PBS buffer as above, detection was achieved with 50 pmol/wellCompound III-labelled anti-GST Ab in Hepes buffer as above, 100 μl finalwell volume. Finally, the plate was washed with PBS buffer, as above and100 μl analar water was added to each well. The plate was read atEx535/Em569 and Ex560/595 in the Biolumin 960 fluorescence microplatereader (Molecular Dynamics Inc.). The results are shown in FIG. 8.

[0213] Detection with Compound III-labelled anti-GST produced a goodsignal of around 10,000 rfu with a corresponding S/N ratio of between101:1 (Ex560!Em595) and 123:1 (Ex535/Em569). Specificity wasdemonstrated using a 40 fold molar excess of a specific competitor, p65which reduced the total signal by approximately 90%. A non-specificcompetitor, casein reduced the total signal by only 25%.

EXAMPLE 15

[0214] Protein:DNA FRET Binding Assay

[0215] 15.1 Preparation of Reagents

[0216] All HPLC purified NF-kB-specific oligonucleotides were obtainedfrom Genosys Biotechnologies Ltd: a coding strand modified with a 5′terminal primary amine (5′ NH₂-GATCTAGGGACTTTCCGCG 3′) and an unmodifiednon-coding strand (5′ ATCCCTGAAAGGCGCCTAG 3′). A 10-fold molar excess ofCy-5-NHS ester dye (Amersham Pharmacia Biotech) was incubated with thecoding strand, in 0.1M sodium bicarbonate buffer (pH9), overnight at 22°C. The following morning the oligonucleotide was ethanol precipitatedand then resuspended in water. The labelled coding strand was purifiedby reverse phase HPLC using a C18 column and a 60% TEAA/acetonitrilegradient. The peak containing labelled oligonucleotide was freeze driedand resuspended in water.

[0217] NF-kB-specific double stranded (ds) DNA was generated byincubating together equimolar amounts of the Cy-5 labelled coding strand(5′ Cy5-GATCTAGG GACTTTCCGCG 3′) and the unmodified non-coding strand(5′ ATCCCTGAAA GGCGCCTAG 3′) in a boiling water bath for 3 minutes andallowing to anneal by cooling over 2 hours.

[0218] NF-kB p65 protein (260 mg) was diluted to 1000 μl in 0.01Mphosphate buffered saline. A 20 fold molar excess of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III), N-hydroxysuccinimide ester (as a 1 mg/ml solution inDMSO) was incubated with the protein at 22° C. with agitation for 2hours (in the dark). The labelled protein was dialysed against threechanges of 0.01M phosphate buffered saline/0.5M NaCl/3 mM EDTA/2 mM DTTat 4° C. (in the dark, 4 hours/fresh buffer).

[0219] 15.2 Binding Assay

[0220] A black microtitre plate (Dynatech) was used for the FRET assay.Compound III-labelled p65 (20 pmol) was incubated in 10 mM Hepes, 0.2 mMsodium acetate, 0.05% NP40, 1 mg/ml BSA and 5 mM DTT with 10 pmol of Cy5labelled NF-kB-specific double stranded (ds) DNA in the presence orabsence of either 200 pmol/well p65 (specific competitor) or casein(non-specific competitor). Both proteins were diluted in the Hepesbuffer, to give a final well volume of 100 μl. Wells containing CompoundIII-labelled p65 only were used as the blank. The plate was incubatedwith agitation for 30 minutes at 22° C. (in the dark) and left to standfor a further 30 minutes. The plate was read at Ex520/Em670 in theBiolumin 960 fluorescence microplate reader (Molecular Dynamics Inc.).The results are shown in FIG. 9.

[0221] A signal of 2000 rfu was obtained in the FRET assay with acorresponding S/N ratio of 4:1. Specificity was demonstrated using a 10fold molar excess of a specific competitor, p65, which reduced the totalsignal by 40%. A non-specific competitor, casein had no effect on thetotal signal.

EXAMPLE 16

[0222] Receptor Ligand Binding Assay Using Fluorescence Polarization

[0223] 16.1 Preparation of Reagents.

[0224] A sample of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III), N-hydroxysuccinimide ester (1 mg) was reacted with 1 mgof telenzepine amine congener* in dimethylsulphoxide in the presence of5% v/v triethylamine. The reaction was allowed to continue for 2 hoursat ambient temperature in the dark. The compound III-telenzepine productwas purified from the starting material by reverse phase HPLC using aC18 column and a 60% water/acetonitrile gradient in the presence of 0.1%trifluoracetic acid. The product peaks were collected, freeze dried inthe dark and resuspended in dimethyl sulphoxide. This was aliquoted andstored frozen at −20° C. in the dark.

[0225] Chinese hamster ovary cells stably expressing the M₁ muscarinicreceptor (CHO M₁ cells) were grown in HAMS F12 media (Sigma) with 10%foetal bovine serum (BRL); 2 mM glutamine; 50 IU/ml penicillin,streptomycin; 125 μg/ml geneticin (Sigma), maintained at 37° C. with 5%CO₂ in a humidified no incubator. The cells were expanded into rollerbottles, purged with 5% CO₂ and left in a roller bottle incubator at 37°C. for 4 days.

[0226] The cells were harvested by scraping into cold phosphate bufferedsaline pH 7.3 (PBS tablets; Sigma) and pelleted by centrifugation at1400 rcf at 4° C. in a MSE Mistral 3000 i centrifuge. Cells wereresuspended in cold 5 mM MgCl₂, 50 mM Tris pH7.5 homogenisation bufferand left on ice for 20 minutes before cell lysis using the Parr celldisruption apparatus (Parr Cat. N°. 4639, 45 ml) using 900 psi ofnitrogen. Any non-disrupted cells were removed by centrifugation at 1400rcf and the supernatant was removed and further centrifuged at 18000 rpmin the Beckman J2-21M/E centrifuge for 20 minutes at 4° C. The resultingpellets were resuspended in cold homogenisation buffer and centrifugedat 18000 rpm as before. The cell membrane pellets were resuspended inapproximately 15 ml of homogenisation buffer, aliquoted and frozen inliquid nitrogen for storage at −70° C.

[0227] 16.2 Binding Assay

[0228] Aliquots of CHO M₁ cell membrane (50 μg) was added to wells of ablack microtitre plate containing a range of dilutions of the M₁muscarinic receptor antagonists atropine and TAC (5000 nM-0.064 nM). Theplate was then measured on the Fluorolite FPM-2™ where backgroundpolarization values were determined (excitation filter 530 nm; emissionfilter 590 nm). After addition of Compound III-telenzepine ligand (4 nMfinal concentration) the plate was sealed and incubated at 22° C. in thedark on a microtitre plate shaker. A final reading was taken after 80minutes. From the polarization data obtained, competition curves weregenerated for both atropine and TAC, using non-linear regression andone-site binding analysis (GraphPad Prism 2.0 data manipulationpackage).

[0229] The curves in FIG. 10 show specific polarization readings plottedagainst log₁₀ molar concentration unlabelled ligand. A specific signalof 190 mP was obtained and the IC₅₀ values determined from thisexperiment were 4.1 nM for atropine and 28 nM for the unlabelled TAC.

1. A compound of formula:

optionally substituted by groups R²-R⁹, wherein groups R⁶, R⁷, R⁸ and R⁹are attached to the rings containing X and Y or, optionally are attachedto atoms of the Z^(a) and Z^(b) ring structures; R² to R⁹ which are thesame or different include —R¹⁰ and —L—R¹⁰ where R¹⁰ is selected fromneutral groups that reduce water solubility, polar groups that increasewater solubility, functional groups that can be used in labellingreactions, reactive groups, electron donating and withdrawing groupsthat shift the absorption and emission wavelengths of the fluorescentmolecule, lipid and hydrocarbon solubilising groups, and L is selectedfrom the group consisting of a straight or branched C₁₋₂₀ alkyl chain, aC₂₋₂₀ monoether or polyether and a C₂₋₂₀ atom chain containing up tofour secondary amide linkages; R¹ is selected from hydrogen, aryl,heteroaryl, cyano, nitro, aldehyde, halogen, hydroxy, amino, quaternaryamino, acetal, ketal, phosphoryl, sulphydryl, water-solubilizing groups,and alkyl groups optionally substituted by amino, C₁-C₄alkyl-substituted amino, quaternary amino, carbonyl including aldehydeand ketone, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl,sulphonate, sulphate, carboxylate, amide, nitro, and groups reactivewith amino, hydroxyl, aldehyde, phosphoryl, or sulphydryl groups; A isselected from O, S and NR¹¹ where R¹¹ is the substituted amino radical:

where R′ is selected from hydrogen, a C₁₋₄ alkyl and aryl and R″ isselected from C₁₋₁₈ alkyl, aryl, heteroaryl, an acyl radical having from2-7 carbon atoms, and a thiocarbamoyl radical; X and Y may be the sameor different and are selected from bis-C₁-C₄ alkyl and C₄-C₅ spiro alkylsubstituted carbon, oxygen, sulphur, selenium, CH═CH, and N—W wherein Nis nitrogen and W is selected from hydrogen, a group —(CH₂)_(n)R¹² wheren is an integer from 1 to 26 and R¹² is selected from hydrogen, amino,aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl,sulphonate, sulphate, carboxylate, substituted amino, quaternary amino,nitro, primary amide, substituted amide, and groups reactive with amino,hydroxyl, carbonyl, phosphoryl, and sulphydryl groups; Z^(a) and Z^(b)each represent a bond or the atoms necessary to complete one, two fusedor three fused aromatic rings each ring having five or six atoms,selected from carbon atoms and, optionally, no more than two oxygen,nitrogen and sulphur atoms; provided that when X and Y are other thancarbon, at least one of R¹-R⁹ comprises a reactive group for covalentreaction with a functional group on a target material or comprises afunctional group for covalent reaction with a reactive group on a targetmaterial, or, when X and Y are different and are selected from O and Se,at least one of R¹-R⁹ is other than hydrogen, methyl, phenyl ornaphthyl.
 2. A compound according to claim 1 wherein R¹⁰ is selectedfrom: hydrogen, halogen, amide, C₁-C₆ alkoxy, nitro, cyano, aryl,heteroaryl, sulphonate, quaternary ammonium, guanidinium, hydroxyl,phosphate, phosphonate, optionally substituted amino, azido, sulphydryl,carboxyl, carbonyl, reactive groups, for example, succinimidyl ester,isothiocyanate, anhydride, haloacetamide, maleimide, sulphonyl halide,phosphoramidite, acid halide, alkylimidate, hydrazide and carbodiimide;and groups reactive with amino, hydroxyl, aldehyde, phosphoryl, orsulphydryl groups.
 3. A compound according to claim 1 wherein R¹ isselected from hydrogen, aryl, heteroaryl, cyano, halogen, alkyl groupsof twenty-six carbon atoms or less and —(CH₂)_(n)Q where 1<n<26 and Q isselected from amino, aldehyde, hydroxyl and groups reactive with amino,hydroxyl, aldehyde, phosphoryl, or sulphydryl groups and R², R³, R⁴ andR⁵ are hydrogen.
 4. A compound according to claim 1 selected from: i)6,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a;3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound I); ii)8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium(Compound II); iii)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III); iv)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,glycinamide (Compound IV); v)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-(2-aminoethylcarboxamide) (Compound V); vi)6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VI); vii)6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VII); viii)6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzothiazolenine-indolenine-[3,2-a]-benzthiazolyl[3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VIII); ix)6,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium(Compound IX); x)6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenium-[3,2-a,3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound X).
 5. A method for producing a compound according to claim 1comprising reacting a compound of formula (A):

optionally substituted by groups R²-R⁹, wherein X, Y, Z^(a), Z^(b) andgroups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are as defined above and Ris methyl or ethyl under conditions suitable for the formation oflinkage A, where A is as hereinbefore defined.
 6. A compound of formula:

optionally substituted by groups R²-R⁹, wherein R⁶, R⁷, R⁸ and R⁹ areattached to the rings containing X and Y or, optionally are attached toatoms of the Z^(a) and Z^(b) ring structures and wherein R¹, R², R³, R⁴,R⁵, R⁶-R⁹, X, Y, Z^(a), Z^(b) and R are hereinbefore defined; providedthat when X and Y are other than carbon, at least one of R¹-R⁹ comprisesa reactive group for covalent reaction with a functional group on atarget material or comprises a functional group for covalent reactionwith a reactive group on a target material, or, when X and Y aredifferent and are selected from O and Se, at least one of R¹-R⁹ is otherthan hydrogen, methyl, phenyl or naphthyl.
 7. A method of impartingfluorescent properties to a non-polar material, the method comprisingthe steps of admixing in the non-polar material a compound as recited inclaim 1, wherein at least one groups R¹ to R⁹ and R¹¹ is an unchargedgroup.
 8. A method of imparting fluorescent properties to a polarmaterial, the method comprising the steps of admixing in the polarmaterial a compound as claimed in claim 1 wherein at least one of groupsR¹ to R⁹ and R¹¹ is selected from the group consisting of charged groupsand polar groups.
 9. A method for imparting fluorescent properties to atarget material, the method comprising the steps of reacting together:i) a target material having at least one functional group selected fromthe group consisting of amino, hydroxyl, phosphoryl, carbonyl andsulphydryl groups; or having at least one reactive group that cancovalently bond with said at least one functional group, and; ii) anamount of the fluorescent compound as claimed in claim 1 wherein atleast one of groups R¹ to R⁹ and R¹¹ is a functional group selected fromthe group consisting of amino, hydroxyl, phosphoryl, carbonyl andsulphydryl; or wherein at least one of groups R¹ to R⁹ and R¹¹ is areactive group that can covalently bond with said at least onefunctional group; for a period of time sufficient to permit said atleast one functional or reactive group of said fluorescent compound tocovalently bond to said at least one reactive or functional group ofsaid target material.
 10. A fluorescent labelled target materialcomprising a target material selected from the group consisting ofantibody, lipid, protein, carbohydrate, nucleotides which contain or arederivatized to contain one or more of an amino, sulphydryl, carbonyl,hydroxyl and carboxyl, phosphate and thiophosphate groups, and oxy ordeoxy polynucleic acids which contain or are derivatized to contain oneor more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl,phosphate and thiophosphate groups, microbial materials, drugs, toxins,particles, plastics or glass surfaces and polymers covalently bound to acompound according to claim
 1. 11. An assay method which comprises: i)binding one component of a specific binding pair with a second componentof said pair, said first component being labelled with a fluorescentdonor dye and said second component being labelled with fluorescent (orquenching) acceptor dye to bring about an energy transfer relationshipbetween said first and second components; and, ii) detecting the bindingof the first and second components by measuring emitted fluorescence;wherein the fluorescent donor dye is a compound according to claim 1.12. A method according to claim 11 wherein said binding assay isselected from the group consisting of immunoassays, nucleic acidhybridisation assays, DNA-protein binding assays, hormone receptorbinding assays and enzyme assays.
 13. An assay method which comprises:i) separating two components which are in an energy transferrelationship, the first component being labelled with a fluorescentdonor dye and the second component being labelled with a fluorescent (orquenching) acceptor dye; and, ii) detecting the presence of the first orthe second component by measuring emitted fluorescence; wherein thefluorescent donor dye is a compound according to claim
 1. 14. A methodaccording to claim 13 wherein the assay is selected from proteolyticenzyme cleavage assays, nuclease cleavage assays and lipase cleavageassays.
 15. A method for the measurement of an analyte in a samplecomprising: i) providing a specific binding partner for the analytewherein the specific binding partner is labelled with a compound asclaimed in claim 1; ii) contacting the analyte to be determined with thespecific binding partner under conditions suitable for binding theanalyte to form an analyte-specific binding partner complex; and, iii)measuring the fluorescence polarization of the analyte-specific bindingpartner complex labelled with the dye to determine the extent ofbinding.
 16. An assay method for the determination of an enzyme in asample, said method comprising: i) providing a substrate for the enzyme,said substrate being labelled with a compound as claimed in claim 1; ii)contacting the labelled substrate with the enzyme under conditionssuitable for initiating the enzymatic reaction; and iii) measuring thefluorescence polarization of the sample to determine the extent ofreaction.
 17. A method for the determination of the sequence of anucleic acid, said method comprising the steps of: i) providing a sampleof said nucleic acid to be sequenced, a primer nucleic acid sequencewhich is complementary to at least a part of said nucleic acid to besequenced, a supply of deoxynucleotides and at least onedideoxynucleotide for terminating the sequencing reaction, and apolymerase; ii) performing nucleic acid chain extension and chaintermination reactions; iii) separating the oligonucleotide fragmentsaccording to size; characterised in that one or more of saiddideoxynucleotides or said primer nucleic acid sequence is labelled witha compound as claimed in claim 1.